Superoxide (O.sub.2.sup.-) is an anionic, one-electron reduced form of molecular oxygen. For a review of the chemistry of superoxide, see Sawyer, D. T., et al. (1981) Acc. Chem. Res. 14:393-400. Following Sawyer, et al., the symbol O.sub.2.sup.- is used herein to denote superoxide. Superoxide is very reactive in aqueous solutions and protic solvents. The rate constant for O.sub.2.sup.- reaction with H.sub.2 O is .apprxeq.1.times.10.sup.7 /mol/sec (Sawyer, et al., 1981). On the other hand, O.sub.2.sup.- is quite stable in aprotic solvents. In general O.sub.2.sup.- behaves as an oxidant, and as a strong nucleophile, depending on the solvent, in particular on the pH or presence of an easily abstractable hydrogen atom. Superoxide also acts as a one-electron reductant of metal ions and complexes.
Superoxide is generated in biological systems as a consequence of aerobic respiration. It is now widely accepted that O.sub.2.sup.- is an important agent in the toxicity of oxygen. All oxygen-metabolizing cells so far examined have been found to contain an enzyme activity, termed superoxide dismutase (SOD) which catalyzes the reaction: O.sub.2.sup.- +O.sub.2.sup.- +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2.
The superoxide dismutases are considered to play a major role in protecting cells and tissues against O.sub.2.sup.-. Recently, the genetic defect of ALS (amyelotropic lateral sclerosis), or Lou Gehrig's Disease, has been determined to be a defective SOD which leads to progressive peripheral nerve degeneration. For a review of SOD, see Fridovich, I. (1974) Adv. Enzymol. 41:35-97. Other neuro-degenerative disorders have been linked to oxidative stress involving, at least in part, O.sub.2.sup.- [Coyle, J. T. et al. (1993) Science 262:689-695]. Superoxide has also been implicated in tissue inflammatory reactions, and in myocardial tissue damage following the clearing of an infarcted vessel with streptokinase or plasminogen activator. Certain oxidative enzymes, e.g., xanthine oxidase, are known to generate O.sub.2.sup.- in the course of oxidizing substrate. The O.sub.2.sup.- generated by action of xanthine oxidase catalyzing an oxidation of a substrate has been measured by a chemiluminescence reaction, Greenlee, L., et al. (1962) Biochemistry 1:779-783. The reaction was shown to activate the chemiluminescence of luminol ( 5-amino-2,3-dihydro-1,4-phthalazinedione) or lucigenin (dimethylbiacridylium) at pH 10.0.
The activity of SOD has been measured by chemiluminescence, using xanthine oxidase to generate O.sub.2.sup.- measured by chemiluminescent light intensity, which is inhibited by the activity of SOD. Huu, T. P., et al. (1984) Anal. Biochem 142:467-475 reported a SOD assay using luminol luminescence and O.sub.2.sup.- generated by xanthine oxidase. A steady state of light intensity was obtained when both xanthine oxidase and SOD were present, the level being dependent on SOD activity when xanthine oxidase was constant. Corbisier, P., et al. (1987) Anal. Biochem. 164:240-247 reported an assay for SOD based on chemiluminescence of lucigenin, using the xanthine oxidase system to generate O.sub.2.sup.-. Pascual, C., et al. (1992) Anal. Lett. 25:837-849 reported an SOD assay using luminol, hydrogen peroxide (H.sub.2 O.sub.2) and horseradish peroxidase (HRP), in which the action of the peroxidase on luminol was reported to generate O.sub.2.sup.- which activated luminol chemiluminescence, which was inhibited by SOD. However, the reaction of lucigenin has been reported to be specific for O.sub.2.sup.- whereas luminol chemiluminescence is induced by a variety of agents [Peters, T. R., et al. (1990) Anal. Biochem. 186:316].
Direct measurement of O.sub.2.sup.- has been difficult because of its short lifetime in aqueous solution, and also because of its reactivity with H.sub.2 O and H.sub.2 O.sub.2 to generate free radical chain reactions which are difficult to quantify with chemiluminescence. Consequently, it has not heretofore been possible to measure O.sub.2.sup.- satisfactorily in aqueous samples, especially in biological materials. [Saran and Bors (1991) Klin. Wochenschrift 69:957-964].
Published methods for measuring O.sub.2.sup.- include direct measurement using electron spin resonance (ESR) and indirect measurement using chemiluminescence or dye reduction. ESR measurement of O.sub.2.sup.- has been disclosed, for example, by Aust, et al. (1993) Toxicol. Appl. Pharmacol. 120:168-178; Samuni et al. (1988) Free Radical Biol. Med. 6:141-148; and Buettner (1990) Free Rad. Res. Comm. 10:11-15. Chemiluminescence measurement using luminol or lucigenin has been disclosed by Archer, et al. (1989) J. Appl. Physiol. 67:1903-1911 and 1912-1921; Henry, et al. (1990) Circulation Res. 67:1453-1461) and Gyllenhammar (1987) J. Immunol. Meth. 97:209-213. Dye reduction methods include reduction of cytochrome c [Fridovich, I., in CRC Handbook of Methods for Oxygen Radical Research (Greenwald, R. A., ed.) pp. 121-122]; reduction tetranitromethane [Bielski, et al. (1985) J. Phys. Chem. Ref. Data 14:1041-1091 and nitro blue tetrazolium (Auclair and Voisin in CRC Handbook, pp. 123-132). ESR measurements are qualitative rather than quantitative due to the nonspecific nature of the interaction of O.sub.2.sup.- with the spin-trapping agents used in these studies. As noted, O.sub.2.sup.- is unstable in aqueous milieu, a factor which has limited the sensitivity of both chemiluminescent and dye reduction methods. The latter are further limited by the relatively low sensitivity of absorbance measurements in the visible spectrum. In addition, other oxidizing or reducing agents present in the sample can reduce the accuracy of the results, and further degrade sensitivity.
Nitric oxide (NO) has recently been shown to act as a short-range cell signal transmitter. For example, NO has been shown to act in vascular endothelium as the endothelium-derived relaxing factor (EDRF), and therefore to be an important component of blood pressure regulation. NO has also been shown to function in the causation of erection in male rats. In addition, NO acts as a signal transmitter in both central and peripheral neurons [Garthwaite, J. (1988) Nature 336:385-388; Radomski, M. W., et al. (1987) J. Pharmacol. 92:639-644]. For a review of NO physiological activities, see Nitric Oxide from L-Arginine: A Bioregulatory System (Moncada, S. and Higgs, E. A. eds) Elsevier, Amsterdam 1990.
Various chemiluminescent reactions have been used to measure NO. Anderson, et al., U.S. Pat. No. 3,659,100 disclosed a method for monitoring air pollutants, including NO. Air containing NO is passed through an adsorption column to remove interfering pollutants, then passed through a limiting orifice where a luminol-H.sub.2 O solution is dispersed as droplets in the gas stream. Luminescence occurs on the surface of the droplets and measured by a photocell. A common method of measuring NO in the gas phase also involves chemiluminescence, but does not employ luminol. Sample gas is mixed with ozone which reacts with NO to yield an activated NO.sub.2 which yields a red chemiluminescence which can be measured by a photon counting system. The method has been adapted to measure NO in aqueous samples by Zafiriou, O. P., et al. (1980) Anal. Chem. 52:1662-1667. The procedure involved stripping the NO from an aqueous solution by flowing a gas stream through the solution, and then measuring NO in the gas phase using the conventional ozone-NO.sub.2 chemiluminescent system. Yet another assay for NO in aqueous medium was described by Mordvintcev, P., et al. (1991) Anal. Biochem. 199:142-146. The authors reacted NO with Fe.sup.2+ -DETC (diethyldithiocarbamate) to form a NO-Fe.sup.2+ -DETC complex. The complex was paramagnetic, allowing detection by electron paramagnetic resonance spectroscopy. Kikuchi, K., et al. (1993) Anal. Chem. 65:1794-1799, reported a chemiluminescent reaction to detect NO, using H.sub.2 O.sub.2 and luminol at neutral pH. H.sub.2 O.sub.2 was reported to react with NO to yield peroxynitrite (ONOO.sup.-) which was said to be the reactant which initiated luminol chemiluminescence. The reported limit of detection was 100 fM, and NO detection in the picomolar range from a perfused rat kidney was described. Interfering luminescence of luminol caused by the presence of hemoglobin and H.sub.2 O.sub.2 was removed by adding desferrioxamine at an optimized concentration. Radi, R., et al. (1993) Biochem J. 290:51-57 reported further characteristics of peroxynitrite-induced luminol chemiluminescence in a nonbiological system using a quenched flow peroxynitrite generating system. The effect of O.sub.2.sup.- in the system was studied by adding potassium superoxide. Peroxynitrite alone was able to induce chemiluminescence of luminol. Both O.sub.2.sup.- and bicarbonate enhanced chemiluminescence suggesting a complex interplay of intermediate reactants affecting the quantitative results. Neither NO nor O.sub.2.sup.- alone were capable of directly inducing significant luminol chemiluminescence.
Other NO assays include trapping of NO by nitroso compounds or nitronyl nitroxides [Joseph, et al. (1993) Biochem. Biophys. Res. Comm. 192:926-934], or by reduced hemoglobin to form an adduct detectable by ESR. Measurement of hemoglobin reduced to Inethemoglobin by NO can be detected spectrophotometrically. A carbon fiber amperometric sensor using metal-porphyrin dispersed in an amorphous silica matrix has been reported [Malinsk et al. (1992) Nature 358:676-678]. Also, an NO sensor using aquocyanobinamide in a silica microprobe has been used to measure NO from tissues and single cells [Shibuki (1990) Neurosci. Res. 9:69-70).
ESR (also called electron paramagnetic resonance) suffers from low and variable spin-trapping efficiency, relatively high cost and many artifacts that hinder analysis [Greenberg et al. (1990) Circ. Res. 67:1446-1452]. The spectrophotometric methods are nonspecific and subject to redox cycling, as in the cytochrome c assay for O.sub.2.sup.- [Archer (1993) Faseb J. 7:349-360]. The ozone assay for NO is reportedly quite specific, however, there are several potential problems caused by acidification of samples and strong reducing conditions prior to measurement [Archer (1993); Chung et al. (1990) J. Pharmacol. Exp. The. 253:614-619]. The use of acids or reducing agents will enhance the chemiluminescence signal, but will tend to cause overestimation of NO levels. In the absence of acids or reducing agents a 10-fold decreased signal can be experienced. There is also potential for oxygen reaction with NO to form nitrates and nitrites. A further disadvantage of the NO/ozone assay is that the equipment for carrying out the assay is relatively costly.
The quantitative assay of O.sub.2.sup.- and NO in aqueous media, especially biological materials, has been extremely difficult to achieve satisfactorily because the extreme lability and reactivity of these compounds and because they are present in minuscule amounts in samples of interest. Chemiluminescence has potential to provide the requisite sensitivity but loss of analyte due to lability and/or side reactions remains an unsolved problem. When NO or O.sub.2.sup.- is generated by a tissue, for example, the true amount present in the tissue remains unknown, since most analyte is believed lost in transit to the cell where the analytical reaction occurs.